Yeasts as a biocontrol for microbial diseases of fruit

ABSTRACT

Compositions for the treatment/prevention of microbial diseases of fruit comprising an effective amount of at least one yeast strain selected from the species Rhodotorula glutinis (Fres.) Harrison and Rhodotorula mucilaginosa (Jorg.) Harrison; optionally in association with one or more agriculturally acceptable carriers or excipients are described, as well as methods involving the same. Compositions may be applied to fruit either pre-harvest or post-harvest with post-harvest treatment being preferred. Microbial diseases of fruit which may be treated include blue mold, grey mold, mucor rot and transit rot of fruit.

BACKGROUND OF THE INVENTION

This invention relates to compositions and methods for thetreatment/prevention of microbial diseases of fruit utilising specificyeast species strains. The invention also relates to biologically purecultures of certain yeast strains and combinations thereof which arecapable of treating/preventing microbial diseases of fruit.

Worldwide, post-harvest losses of fruits and vegetables have beenestimated to be up to 50 percent of harvested crop and much of this isdue to microbial diseases such as rots. In the USA, where more attentionhas been directed towards the post-harvest problem, losses of fruits andvegetables due to post-harvest spoilage still amount to about 24%.Pre-harvest losses of fruits and vegetables are also significant.

Fungicides, which are the principal means of controlling post-harvestrots and other microbial diseases, have recently come under security byhealth authorities in may countries in view of the potential effect ofresidues on consumers. According to a US National Science Academy Report(1987), as a class, fungicides account for 60% of the oncogenic riskamong all the pesticides used for food produce. Furthermore, the USEnvironmental Protection Authority has recently proposed a ban on theuse of ethylene bis-dithiocarbamates (EBDCs) on food crops. Aboutone-third of all fruit and vegetables in the USA are treated with EBDCs.Benomyl, a fungicide widely used to protect apples and pears from attackby blue and grey mould, was also discontinued for post-harvest use in1989.

Contamination of the environment by fungicides and risks to the healthof farmers are other causes for concern.

A number of organisms are associated with microbial diseases of fruit.

At least 11 species of Penicillium have been isolated from pome fruitsnaturally infected with blue mould but P. expansum is by far the mostcommon and economically important species. Blue mould, also known assoft rot and wet rot, is the most important post-harvest disease ofapples and is also important on pears.

Grey mould is the most important post-harvest disease of pears and issecond to blue mould in importance on apples. It is caused by the fungusBotrytis cinerea Pers. Also known as cluster rot or nest rot, grey mouldcan cause large losses because of its ability to spread from infected toadjacent healthy fruit during storage. The disease develops more rapidlyat cold storage temperature than any other post-harvest decay exceptMucor rot.

Mucor rot is caused primarily by Mucor piriformis E. Fischer. Mucor rotoccurs less consistently than blue mould and grey mould, although inspecial situations it can cause several losses of apples and pears.

All of these rots are worldwide in occurrence, and affect many types offruit.

SUMMARY OF THE INVENTION

The traditional method of control of these rots and other microbialdiseases is to treat fruit after harvest and before storage withfungicides as discussed above. Benzimidazole and dicarboximidefungicides applied as post-harvest dips, drenches or line sprays areeffective against both blue and grey mould. However, fungicide-tolerantstrains which are present in most packing-houses, reduce theireffectiveness and additional fungicides, e.g., imazalil, have to be usedin combination to improve control. This, together with the need forreduced chemical usage on food crops has created a need to develop othermethods of controlling post-harvest diseases.

In 1987, Janisiewicz demonstrated that blue mould on apples can becontrolled with an antagonistic bacterium and a yeast and in 1988 alsoreported that two antagonistic microorganisms, viz Acremonium breve anda Pseudomonas sp., when tested as mixtures on wounded apples inhibitedthe development of lesions caused by P. expansum and B. cinerea(Janisiewicz 1987; Janisiewicz 1988). In 1988, Wisniewski et al.demonstrated the effectiveness of yeast, Debaryomyces hansenii tocontrol post-harvest decay of apples caused by B. cinerea and, in 1990Chalutz and Wilson reported that D. hansenii was able to inhibit theincidence of green and blue mould and sour rot of several citrus fruitcultivars. Investigations with isolates of D. Hansenii have indicatedthat this antagonist is not particularly effective in controlling blueand green mould of pome and other fruit. The fungus A. breve previouslyproposed for the treatment of microbial disease in fruit is a slowgrowing fungus which is difficult to produce in large quantities forcommercial application.

It is very unpredictable whether particular yeast species would beeffective in the treatment/prevention of microbial disease in fruit.Where a yeast strain such as D. hansenii is at least partially effectivein protecting fruit from microbial diseases, other yeast strains fromspecies capable of growing on fruit may be totally ineffective intreating/preventing microbial disease.

As a result of painstaking investigations by the applicants, it hassurprisingly been found that yeast strains selected from the speciesRhodotorula glutinis (Fres.) Harrison and, Rhodotorula mucilaginosa(Jorg.) Harrison, and combinations of one or more such strains areeffective in the treatment/prevention of microbial diseases of fruit.

In accordance with the first aspect of this invention there is provideda composition for the treatment/prevention of microbial diseases offruit comprising an effective amount of at least one yeast strainselected from the species Rhodotorula glutinis (Fres.) Harrison and,Rhodotorula mucilaginosa (Jorg.) Harrison, optionally in associationwith one or more agriculturally acceptable carriers or excipients.

Yeast strains of the above species are effective in thetreatment/prevention of microbial disease of fruit. Particularlypreferred strains are Rhodotorula glutinis (Fres.) Harrison strain H10deposited with the Austrialian Government Anaytical Laboratory underAccession No. 92/15655; and Rhodotorula mucilaginosa (Jorg.) Harrisonstrain D9 deposited with the Australian Government Analytical Laboratoryunder Accession No. 92/15656. and derivatives/mutants of said straineffective in the treatment/prevention of microbial disease of fruit.

While this invention is specifically described hereafter with referenceto Strains H10 and D9, it is to be understood that the invention is notso limited and extends to any yeast strain of the species Rhodotorulaglutinis (Fres.) Harrison, and Rhodotorula mucilaginosa (Jorg.)Harrison; or mutants/derivatives thereof, optionally in association withone or more agriculturally acceptable carriers or excipients which haveactivity in the treatment/prevention of microbial diseases of fruits. Asdescribed hereinafter, we have devised rapid and routine assays whereyeast strains can be screened for the treatment/prevention of microbialdiseases of fruit. For example, fruit such as apples or pears can bewounded and infected with a microorganism which gives rise to amicrobial disease (such as, grey mould, blue mould, transit rot, mucorrot or the like). The fruit may then be treated with a composition of ayeast strain and after a few days incubation at 20° to 30° C. it can bevisually scanned to see whether or not the onset of microbial diseasehas been prevented by a candidate yeast strain.

Yeast strains may be readily isolated and cultivated according tostandard procedures (for example as described by Phaff, Miller and Mark,The Life of Yeasts, 2nd Edition, Harvard University Press 1978; andDevenport, R. R., Outline Guide to Media and Methods for Studying Yeastsand Yeast Like Organisms, in Biology and Activity of Yeasts, A. P.London, 1980, both of which are incorporated herein by reference), suchas growth on nutrient agar (for example, potato dextrose agar). Yeaststrains can be readily typed according to standard procedures (such asdescribed in Yeasts: Characteristics and Identification by Barnet et al.[1983] Cambridge University Press) to ascertain whether they belong tothe species Rhodotorula glutinis (Fres.) Harrison, Rhodotorulamucilaginosa (Jorg.) Harrison, Candida parapsilosis (Ashf.) Langeron andTalice, and Candida guilliermondii (Cast.) Langeron.

This invention extends to derivatives/mutants of the strains H10, and D9which may be prepared according to standard microbiological methods,such as chemical mutation with mutagenic agents (such asnitrosoguanidine, ethanemethylsulphonate and the like), radiation withan energy source (such as UV radiation, infrared radiation, irradiationwith α, β, or γ particles from a radiation source, and the like),screening for spontaneous mutants, recombinant DNA techniques and othermethodologies as are well known in the art. Derivatives/mutants whichare derived from the strains H10, and D9 may differ from the parentstrains in respect of one or more of morphology, biochemicalcharacteristics, growth characteristics and the like.Derivatives/mutants which are effective in the treatment/prevention ofmicrobial diseases of fruit are embraced by the present invention. Asused herein the terms "derivatives" and "mutants" are synonymous andrefer to any strain derived by whatsoever means from the strains H10, D9and D20.

Compositions may be provided in any of the standard forms known in theart, such as an aqueous suspension, slurry, paste, concentrate orlyophilised form. Lyophilised cultures may be readily re-suspended inaqueous solutions for application to fruit.

Compositions of yeast strains may be in association with one or moreagriculturally acceptable carriers or excipients. The term "carriers"includes water, buffer solutions, carbohydrate containing solutions,saline solutions and any other material suitable for the maintenance ofyeast strains and the like as are well known in the art. The term"excipient" refers to conventional additives, such as surfactants,antioxidants, nutrients, fungicides and the like as are well known inthe art.

Compositions of this invention may additionally comprise a source ofcalcium, such as calcium chloride or other non-toxic calcium source (forexample and proprietary calcium sources such as Stopit (PhosynInternational). It has surprisingly been found that compositionscontaining an effective amount of at least one yeast strain selectedfrom the species Rhodotorula glutinis (Fres.) Harrison, Rhodotorulamucilaginosa (Jorg.) Harrison; in association with a source of calciumshow synergistic/potentiated effects in the treatment/control ofmicrobial diseases. The potentiating effect that calcium has one theyeast strains of this invention is not well understood. Calcium may beprovided in an amount from 0.1 to 50% (v/v).

Compositions may additionally comprise anti-scald agents, such asdiphenylamine or ethoxyquin and other compounds commonly used in the artfor the treatment of fruit.

Additionally, compositions of this invention may include a fungicide,such as imazalil. Given the microbial inhibiting effects of the yeastspecies of this invention less fungicide than conventionally used wouldbe required.

Compositions of this invention have particular application to thepost-harvest treatment of fruit. Harvested fruit is readily amenable totreatment with the compositions of this invention according to standardprocedures for the application of compositions, such as pesticides orfungicides, to fruit. Notwithstanding this, the compositions of thisinvention may be applied to fruit pre-harvest again according tostandard procedures.

Preferred fruits which may be treated in accordance with this inventioninclude pome fruits (such as apples and pears), stone fruits (such aspeaches, nectarines, apricots, plums, cherries), citrus fruits (such aslemons, oranges, mandarines and limes) and grapes.

Microbial diseases of fruit which may be treated in accordance with thisinvention include blue mould, grey mould and Mucor rot of fruit as wellas other microbial diseases which affect fruit. For reasons which arenot well understood, yeast strains selected from the species Rhodotorulaglutinis (Fres.) Harrison and, Rhodotorula mucilaginosa (Jorg.) Harrisonare effective in the treatment/prevention of a wide range of microbialdiseases of fruit.

The compositions of this invention are generally provided in an amounteffective to treat/prevent microbial disease of fruit. This amount willvary depending upon the activity of the yeast strain, the type of fruitbeing treated, age of the fruit, and like factors. Generally, butwithout limiting this invention, compositions of this invention maycomprise from 1×4¹⁰ cells/ml to 1×10¹² cells/ml. Preferably,compositions comprise in excess of 1×10⁷ cells/ml. The actual amounts ofone or more yeast strains is not important as long as the amount issufficient to treat/prevent microbial disease.

In accordance with a further aspect of this invention there is provideda biologically pure culture of Rhodotorula glutinis (Fres.) Harrisonstrain H10 deposited with the Australian Government AnalyticalLaboratory under Accession No.

In another aspect of the invention there is provided a biologically pureculture of Rhodotorula mucilaginosa (Jorg.) Harrison strain D9 depositedwith the Australian Government Analytical Laboratory under Accession No.

In another aspect of this invention there is provided a culturecomprising a mixture of at least two of the strains H10 and D9.

The biologically pure cultures may be provided in any form as is wellknown in the art, such as an aqueous suspension, concentrate, paste,slurry or lyophilised form.

In a further aspect of this invention there is provided a method for thetreatment/prevention of microbial disease of fruit comprising applyingto said fruit an effective amount of a composition comprising at leastone yeast strain selected from the species Rhodotorula glutinis (Fres.)Harrison and, Rhodotorula mucilaginosa (Jorg.) Harrison, optionally inassociation with one or more agriculturally acceptable carriers orexcipients.

The compositions for use in the method of this invention are aspreviously described herein.

Preferred yeast strains are selected from Rhodotorula glutinis (Fres.)Harrison strain H10 deposited with the Australian Government AnayticalLaboratory under Accession No. 92/15655; and Rhodotorula mucilaginosa(Jorg.) Harrison strain D9 deposited with the Australian GovernmentAnalytical Laboratory under Accession No. 92/15656; orderivatives/mutants of said strains effective in thetreatment/prevention of microbial disease of fruit.

In the method of this invention, compositions of yeast strains may beapplied to fruit by methods well known in the art, for example byspraying dipping, drenching or as a mist.

Compositions comprising one or more yeast strains are administered fruitpre-harvest, or post-harvest in an effective microbial inhibitingamount. Generally, but without restricting this invention, such anamount may comprise from 1×10⁴ cells/ml to 1×10¹² cells/ml. Preferably,compositions will comprise at least about 1×10⁹ cells/ml, although thisamount may vary according to factors well known in the art. Thisinvention is not limited to a specific concentration of yeast cells, butrather an amount effective to inhibit pathogenic microorganisms whichcause disease in fruit.

The method of this invention may be practiced post-harvest, that is,after the fruit has been harvested, and/or may be conducted pre-harvest.

The method of this invention is particularly effective in the treatmentblue mould, grey mould, green mould, sour rot and Mucor rot of fruit aswell as other microbial diseases of fruit. In particular, this aspect ofthe invention is effective against the organisms P. expansum, Botrytiscinerea and Mucor piriformis.

Fruit which may be treated in accordance with this invention includespome fruit (such as apples and pears), stone fruit (such as peaches,nectarines, apricots, plus and cherries), citrus fruit (such as oranges,mandarins, lemons and limes) and grapes as previously described. Appleswhich may be treated in accordance with this invention include GrannySmith, Red and Golden Delicious, Jonathan, Gala and strains thereof,Fuji, Newton, Macintosh and other well known apple strains. Examples ofpears include Packham's Triumph, William's Bon Chretian and Beurre Bosc.

Fruit treated in accordance with the methods of this invention may bestored at standard fruit temperature storage, such as 0° C., 4° C. androom temperature free of the effects of microbial infection or withreduced susceptibility to infection. This is most important as fruit,such as apples and pears, may be stored for a significant time periodbefore sale or use. By way of example, fruit may be stored attemperatures of about 0° or 4° C. up to at least 12 months withoutspoilage. Fruit treated in accordance with this invention may also bestored in controlled atmosphere (such as 2.5% oxygen and 2.5% carbondioxide) again without microbial infection, or control of microbialinfection.

In a further aspect of this invention there is provided fruit which hasbeen treated with an effective amount of a composition comprising aneffective amount of at least one yeast strain selected from the speciesRhodotorula glutinis (Fres.) Harrison, and Rhodotorula mucilaginosa(Jorg.) Harrison. In particular, this aspect of the invention providesfruit which has been treated with a composition comprising Rhodotorulaglutinis (Fres.) Harrison strain H10 deposited with the AustralianGovernment Anaytical Laboratory under Accession No. 92/15655; andRhodotorula mucilaginosa (Jorg.) Harrison strain D9 deposited with theAustralian Government Analytical Laboratory under Accession No.92/15656; or derivatives/mutants thereof as previously described. Suchfruit is resistant to the effects of microbial disease of fruit and thusmay be stored for extended time periods and handled without problems ofmicrobial disease infection.

Fruit treated according to this invention includes pome fruit, stonefruit, citrus fruit and grapes. Examples of such fruits include apples,pears, peaches, nectarines, apricots, plums, cherries, grapes, oranges,madarins, lemons and limes.

The novel yeast strains hereafter described have unexpected and verypotent activity in the treatment/prevention of microbial disease offruit.

This invention will now be described with reference to the followingnon-limiting Figure and Examples.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the effect of strain D9 on blue mould developments onwounded Golden Delicious apples, photographed 8 days after inoculationwith Penicillium expansum. Treatment 6B was inoculated with P. expansumonly.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1

A. Isolation of Yeast Strains

Yeast strains were isolated according to standard procedures (forexample as described by Phaff, Miller and Mrak, The Life of Yeasts, 2ndEdition, Harvard University Press, 1978; and Devenport, R. R., OutlineGuide to Media and Methods for Studying Yeasts and Yeast Like Organisms,in Biology and Activity of Yeasts, A. P., London, 1980, both of whichare incorporated herein by reference) utilising, for example, variousagars containing yeast nutrients and optionally selective agents such asone or more antibiotics.

A large number of yeast strains were isolated from sources such as thesurface of fruit, fruit storage crates, butter and other dairy products.Yeast strains were then tested for activity in preventing microbialdiseases of fruit according to the routine assay set forth in part Cbelow.

A significant number of the yeast strains isolated (about 50) had no ornegligible activity in preventing microbial fruit diseases such as bluemould, grey mould, Mucor rot and transit rot.

Strains from yeast species consistently exhibited activity intreating/preventing microbial diseases of fruit, these species beingRhodotorula glutinis (Fres.) Harrison, and Rhodotorula mucilaginosa(Jorg.) Harrison. It is currently unclear why these species are activein treating/preventing microbial fruit diseases. The isollation andcharacterisation of these yeast strains designated H10, and D9.

Strain H10

Strain H10 was isolated by pressingdichloran-chloramphenicol-rose-bengal agar (King et al. 1979) platesagainst the inner wall of wooden fruit boxes, used as containers on acommercial orchard. Exposed plates were incubated for 5 days at roomtemperature and the bacterial and yeast colonies that developed weretransfered to potato-dextrose agar (PDA-Oxoid, Unipath Ltd.,Basingstoke, Hampshire, England) plates. After cleaning out byre-streaking a few times, clean isolates were transfered to PDA slopesand maintained at 4° C.

Strain D9

This strain was isolated from spoilt butter which was diluted and platedon oxytetracycline-glucose-yeast extract agar and incubated at 25° C.for 5 days. Resulting yeast colonies were isolated and cleaned asdescribed for H10.

Strain D20 (comparative)

The procedure used was identical to D9 but the source was culturedbutter milk.

B. Identification of Strains

Yeast species were identified according to standard procedures, such asdescribed in "Yeasts: Characteristics and Identification" by Barnett etal. (1983) Cambridge University Press.

The representative yeast strains H10, and D9 were identified by theCentralbureau voor Schimmelcultures, Delft, The Netherlands, as follows:

Strain H10--Rhodotorula glutinis (Fres.) Harrison;

Strain D9--Rhodotorula mucilaginosa (Jorg.) Harrison and

Comparative Strain D20 is C. guilliermondii (Cast.) Langeron and Guerra.(teleomorph Pichia guilliermondii Wickerman).

C. Routine Testing Procedures for Effectiveness of Strains AgainstMicrobial Pathogens

(i) A standard method for effectiveness was established using apples,although of course, other fruits could be used.

For ease of reference hereafter, yeast strains selected for screening mbe referred to as "antagonists" or "antagonistic strains".

In initial screening tests, apple wounds, 3 mm deep and 3 mm indiameter, were inoculated with 30 μl of an aqueous suspension of theantagonistic strain having a concentration 1×10⁷ to 1×10⁸ cells/ml.After hours, the wounds were inoculated with 20 μl of an aqueoussuspension of spores of the pathogen, having a concentration of 1×10⁴ to1×10⁵ spores/ml. Control apples were inoculated with the pathogen only.Inoculated apples were incubated at 24° C. and lesion diameters weremeasured after 6, 7 or 8 days. Each apple constituted a single replicateand each treatment was replicated 5, 6 or 10 times.

Within a few days an assessment of effectiveness in preventing microbialdisease can be made.

(ii) After initial screening as described above, selected antagonistswere tested further by dipping fruit in a suspension of the antagonists.Fruit was first dipped in the antagonist suspension and after 2 hoursdipped in a suspension of the pathogesn. Alternatively, fruit was dippedin a suspension of the antagonist which was loaded with spores of thepathogen under test. Dipping time was 30 seconds. In such experimentsthe antagonists were used at a concentration of 1×10⁸ to 1×10⁹ cells/mland the pathogen at 1×10⁴ spores/ml. Fruit was washed, wiped dry andthen wounded in 10 places by puncturing with a nail passed through arubber stopper. The wounds were conical in shape with a diameter of 2 mmand 4 mm deep. Treated and control fruit were incubated at 24° C. andthe percentage of wounds that developed into lesions was recorded after6, 7 or 8 days.

Both of the above tests are highly artificial, representing a very highpathogen concentration as well as level of wounding

D. Storage of Cultures

Strains were stored as freeze dried cultures in the dark at roomtemperature. For routine work, cultures are stored on PDA slopes at 4°C. and replaced every 3-4 months with freeze dried cultures. No loss ofactivity has been observed with cultures stored in this way. Viablecultures of the strains H10, D9 and D20 have been deposited in theInstitute of Plant Sciences Collection at Burnley as well as with theAustralian Government Analytical Laboratory as described herein.

E. Cultivation of Strains

For test on antagonistic activity, the strains are normally grown on PDAat 24° C. and 3 to 6 day old cultures were washed off the plates andsuspended in sterile distilled water.

When large quantities are required, the strains are cultured in variousliquid media but the preferred medium is nutrient broth supplementedwith yeast extract and dextrose (NYDB--Nutrient Broth 0.8%, YeastExtract 0.5%; Dextrose 1.0%). The pH of this medium was 6.6. Liquidcultures were grown in conventional flasks on an orbital shaker (60 rpm)at room temperature for 48 hours, after which the medium was centrifuged(8000 g, 30 min) and the pellet washed with sterile distilled water andresuspended in the same medium. Final concentrations were adjusted bydirect cell count using a haemocytometer.

EXAMPLE 2 Effectiveness of strain D9 against blue mould (P. expansum)and grey mould (B. cinerea)

In the experiment, strain D9 was tested (according to Example 1(c)(i))together with four other strains (E.a--Enterobacter aerogenes,C.a--Cryptococcus albidus a standard yeast strain; TV3--Trichodermaviridie, a common fungus; B.s--Bacillus subtilis, a common bacterium)for its effectiveness against blue and grey mould. There were 5replicates per treatment and lesions were recorded after 8 days. Resultsare shown in Table 1 below. Control apples were only inoculated withpathogen.

                  TABLE 1                                                         ______________________________________                                        Effectiveness of 6 Antagonistic Strains Against Blue Mould                    (P. expansum) and Grey Mould (B. cinerea) of Apple                                       Mean diameter (mm) of lesion                                                  8 days after inoculation                                           Antagonist   P. expansum B. cinerea                                           ______________________________________                                        E.a          27.2        4.8                                                  C.a.         17.8        5.9                                                  D9           6.1         4.2                                                  TV3          33.2        0                                                    B.s          38.5        30.3                                                 Control      40.9        32.1                                                 ______________________________________                                    

Table 1 clearly shows that the strain D9 is effective in the control ofboth blue and grey mould. In contrast, none of the other strains testedwas considered to be effective against blue mould, while someeffectiveness was exhibited against grey mould.

FIG. 1 shows the effectiveness of the strain D9 (designated row 3b) whencompared with control apples inoculated only with the pathogen P.expansum.

EXAMPLE 3 Effectiveness of Strains H10, D9 and D20 (comparative) AgainstGrey Mould (B. cinerea) of Apple

Eleven antagonists were tested in this experiment including the selectedtest strains H10, D9 and D20. Comparative strains were selectedrandomly. These strains were: isolated from the surface of peach (A6a);Trichoderma verdi (TV3); Debaryomyces hansenii a yeast strain previouslydescribed to be effective for the treatment of fungal disease of fruit(Hu9); a random yeast isolated from the surface of apricots (Ap2); ayeast isolated from a fruit crate (H9); and a yeast isolated from anapricot surface (A4). Control fruit was inoculated only with pathogen.There were 6 replicates per treatment and lesion diameters were measured7 days after inoculation. Results (Table 3) demonstrated theeffectiveness of the three strains H10, D9 and D20, against grey mould.Some of the other strains investigated were also effective. Strain H9was found to belong to the yeast species Geotrichum klebahnii. Thisstrain is on deposit with the Institute of Plant Sciences Collection atBurnley, Victoria, Australia.

                  TABLE 2                                                         ______________________________________                                        Effectiveness of 11 Antagonistic Strains Against                              Grey Mould (B. cinerea) of Apple                                                          Mean lesion diameter (mm)                                         Antagonist  7 days after inoculation                                          ______________________________________                                        D9          2.5                                                               C.a.        1.3                                                               H10         0                                                                 A6a         0                                                                 TV3         6.7                                                               Hu9         8.7                                                               H9          0                                                                 Ap2         5.3                                                               D20         0                                                                 A9          0                                                                 A4          0                                                                 Control     30.1                                                              ______________________________________                                    

EXAMPLE 4 Effect of Varying Concentrations of Antagonists and Pathogenon Lesion Development

To determine the minimum concentration of antagonist that willcompletely inhibit lesion development at a given concentration of thepathogen, a number of experiments were carried out using varyingconcentrations of the antagonists (2×10⁶ -1.2×10⁹ cells/ml) andpathogens (0.7×10³ -1.2×10⁶ spores/ml). Fruit was wounded and inoculatedwith the antagonist first and then with the pathogen and lesiondiameters were recorded after incubation at 24° C. for 7 days.

Results of one experiment are shown in Table 3. It is evident that about1×10⁹ cells/ml of the antagonist are required to inhibit lesiondevelopment at a pathogen concentration of 1×10⁴ spores/ml under thehighly artificial test conditions. Under conventional conditions ofapple storage our results indicate that yeast cells in excess of about1×10⁷ should be effective.

                  TABLE 3                                                         ______________________________________                                        Effect of Varying Concentrations of Strain D9 on                              Grey Mould (B. cinerea) Development in Apple.                                 Mean Lesion Diameter (mm).                                                    Concentra-                                                                             Concentration of B. cinerea                                          tion of D9                                                                             (spores/ml)                                                          (Cells/ml)                                                                             1.21 × 10.sup.6                                                                   1.21 × 10.sup.5                                                                   1.21 × 10.sup.4                                                                 1.21 × 10.sup.3                    ______________________________________                                        1.17 × 10.sup.9                                                                  29.0      13.5      0       0                                         0.6 × 10.sup.9                                                                  35.6      20.5      0       0                                        0.30 × 10.sup.9                                                                  36.1      24.5      0       0                                        0.15 × 10.sup.9                                                                  42.1      21.8      8.3     0                                        0.17 × 10.sup.8                                                                  36.4      23.3      12.9    0                                        SDW-check                                                                              44.6      37.4      25.1    7.9                                      ______________________________________                                    

EXAMPLE 5 Effectiveness of Strains H10 and D9 Against Grey Mould (B.cinerea) of Apple

In this experiment, strains H10 and D9 were tested for activity againstgrey mould together with two other yeast (H9 and C.a.). Fruit (cv.Granny Smith) was first dipped in a suspension of the antagonist and 2hours later dipped in a suspension of the pathogen (according to Example1(C)(ii)). Control fruit was dipped only in the pathogen suspension.There were 12 fruit per treatment. Results were observed after 9 daysafter inoculation and none of the fruit treated with the antagonistsdeveloped any lesions, whereas the 12 control fruit had 21 lesions.

EXAMPLE 6 Effectiveness of Strain H10 Against Blue Mould of Apple

Strain H10 and two other strains (C.a. and H9) were tested in this trialon Granny Smith Apples according to the method of Example 1(C)(ii).There were 12 fruit per treatment and lesions were recorded 7 days aftertreatment. Again, none of fruit treated with the antagonists developedlesions but the 12 control fruit had 54 lesions (45% infection).

EXAMPLE 7 Effectiveness of H10, D9 and D20 (comparative) Against MucorRot of Apple

This test was performed on Granny Smith apples using 10 fruit pertreatment. The strains H10, D9 and D20 were compared with strains C.a.and H9 as previously described, and then random isolates D27. A standardchemical treatment which contained iprodione, benomyl and imazalil(0.068%) was included for comparison. Lesions were recorded afterincubating fruit for 7 days at 24° C.

Results indicated that strain D20 was as good as the chemical treatment,while H10 and D9 give significant control (Table 6). Strains H9, D27 andC.a. also showed some degree of effectiveness.

                  TABLE 4                                                         ______________________________________                                        Effectiveness of Strains H10, D9 and D20 and Two Other                        Strains Against Mucor Rot of Apple                                            Treatment    Percentage Infection                                             ______________________________________                                        H9           10                                                               H10          12                                                               D9           12                                                               D20           2                                                               D27          14                                                               C.a          14                                                               Chemical      4                                                               Control      43                                                               ______________________________________                                    

In the experiments described so far, fruit was dipped one at a time in200-300 ml of the antagonist and/or pathogen suspension. The twoexamples given below describe experiments in which losts of 10 fruitwere dipped simultaneously in 2 liters of the appropriate suspension.

The effect of adding calcium to the antagonistic suspension on rotcontrol was also investigated, since calcium has been described asplaying an important role in fruit softening and other aspects of fruitquality (Pooviah et al. 1990).

EXAMPLE 8 Effect of Strains H10, With and Without Added CalciumChloride, on Blue Mould Development in Apples and Pears

In this experiment, fruit were wounded and then dipped in a suspensionof the antagonist (1×10⁹ cells/ml), with and without calcium chloride(2%), and containing 1×10⁴ spores/ml of P. expansum. Control treatmentsconsisted of a solution of calcium chloride and distilled water bothhaving the same concentration of the pathogen. Fruit was incubated at24° C. and percentage of wounds developing into lesions were recordedafter 6 days.

Results (Table 5) indicated that addition of 2% calcium chloride to theantagonistic suspension unexpectedly improved the effectiveness of theantagonist in controlling blue mould in both apples and pears. Thiseffect is synergistic being greater than the sum of H10 protection andcalcium protection. In the case of pears, it also appeared thattreatment with calcium chloride alone gives significant control of bluemould. It is also evident that fruit can be dipped in bulk in theantagonist suspension and still obtain good control of infection.

                  TABLE 5                                                         ______________________________________                                        Effectiveness of Strain H10, With and Without Added Calcium                   Chloride, Against Blue Mould of Apple and Pear                                                Percentage Infection                                          Treatment         Apples   Pears                                              ______________________________________                                        H10 only          13       10                                                 H10 + calcium chloride                                                                           7        5                                                 Calcium chloride only                                                                           94       27                                                 Control           98       61                                                 LSD (p = 0.05)     9       23                                                 ______________________________________                                    

EXAMPLE 9 Effect of Strain H10, With and Without Calcium Chloride, onGrey Mould Development in Apples and Pears

The procedures used were identical to those in the previous example.

Results (Table 6) show that the addition of calcium chloride to theantagonist suspension improves the control of grey mould. Furthermore,calcium chloride alone has a significant effect on grey moulddevelopment but only in pears.

                  TABLE 6                                                         ______________________________________                                        Effect of Calcium Chloride on the Activity of Strain H10                      Against Grey Mould of Apple and Pear                                                          Percentage Infection                                          Treatment         Apples   Pears                                              ______________________________________                                        H10 only          52       33                                                 H10 + calcium chloride                                                                          20       20                                                 Calcium chloride only                                                                           95       52                                                 Control           100      82                                                 LSD (p = 0.05)    17       29                                                 ______________________________________                                    

EXAMPLE 10 Effect of DPA on the Antagonistic Activity of the SelectedStrains

DPA, diphenyl amine, is used as an antiscald agent on some varieties ofapples and pears which tend to brown after long cold storage. The objectof this experiment was to determine the effect of DPA on theantagonistic activity of the strains H10, D9 and D20 when used incombination. Results are given below.

                  TABLE 7                                                         ______________________________________                                        Effect of DPA on the Activity 3 Antagonists                                   Against Blue Mould of Apples                                                               Percentage Infection                                             Treatment    after 7 days                                                     ______________________________________                                        H10          4                                                                H10 + DPA    3                                                                D9 only      2                                                                D9 + DPA     3                                                                C.a only     2                                                                C.a. + DPA   3                                                                DPA only     26                                                               Control      38                                                               ______________________________________                                    

Results show that DPA is compatible with the antagonists tested.In-vitro tests also show that DPA at 2,000 ppm (the highest recommendedrate) has no effect on the growth of the antagonists.

EXAMPLE 11 Effectiveness of Strains D9, H10 and D27 Against Blue Mouldof Pear Compared with Fungicide Treatment

The three antagonists were tested at a concentration of 1×10⁹ cells/ml.The pathogen concentration was 1×10⁴ spores/ml.

DPA was added to the appropriate suspension to a final concentration of1500 ppm. The chemical treatment consisted of iprodione, benomyl andDPA. There were 10 fruit per treatment and 10 wounds per fruit. Treatedand control fruit were incubated for 7 days at 24° C. before lesionswere recorded.

Results are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                        Effectiveness of 3 Antagonists Against Blue Mould of Pear,                    Compared with Fungicide Treatment                                             Treatment     Percentage Infection                                            ______________________________________                                        D9 only       17                                                              D9 + DPA      15                                                              H10 only       7                                                              H10 + DPA     11                                                              D27 only      34                                                              D27 + DPA     26                                                              Fungicide     30                                                              Control       74                                                              LSD (p = 0.05)                                                                              21                                                              ______________________________________                                    

EXAMPLE 12 Effectiveness of Strains H10, D9 and D20 (comparative)Against Mucor Rot of Apple

The concentrations of the antagonists and the pathogen used were thesame as in the previous example. In this test, the effect of adding 2%calcium chloride to the antagonistic suspension on the control of bluemould was also investigated. The chemical treatment consisted ofiprodione, benomyl and imazalil at the same concentrations as previouslyindicated. Fruit was incubated at 24° C. and results were recorded 8days after treatments were applied as set forth in Table 9.

                  TABLE 9                                                         ______________________________________                                        Effectiveness of Strains H10, D9 and D20, With and Without                    Calcium Chloride, Against Mucor Rot of Apple,                                 Compared with Fungicide Treatment                                             Treatment        Percentage Infection                                         ______________________________________                                        H10 only         7                                                            H10 + calcium chloride                                                                         0                                                            D9 only          4                                                            D9 + calcium chloride                                                                          0                                                            D20 only         7                                                            D20 + calcium chloride                                                                         0                                                            Chemical         2                                                            Control          79                                                           ______________________________________                                    

These experiments (Examples 11 and 12) indicate that the three selectedstrains control post-harvest rots of pome fruit as effectively as thecurrently used chemical treatment. The strains are also compatible withDPA and the addition of calcium chloride to the antagonistic dipimproves control in a synergistic manner.

EXAMPLE 13

A number of investigations were carried out to determine if the selectedantagonists would be active after holding treated fruit at 0°-1° C., theusual temperature at which fruit, such as pome fruit, is stored.

A. Effectiveness of 5 strains against blue mould of apple after holdingtreated fruit at 1° C. for 9 days

The antagonists used were strains H], H10, D9, D20 and C.a. Fruit waswounded and dipped in a suspension of the antagonist loaded with sporesof P. expansum, held at 1° C. for 9 days and then transfered to 24° C.for 7 days. A fungicide treatment was included for comparison. Resultsare shown in Table 10.

                  TABLE 10                                                        ______________________________________                                        Effectiveness of 4 Antagonistic Strains Against Blue Mould of                 Apple, After Holding Treated Fruit for 9 Days at 1° C.                              Percentage Infection                                             Antagonist   after 1 week at 24° C.                                    ______________________________________                                        H9           24                                                               H10          9                                                                D9           6                                                                D20          5                                                                Chemical     2                                                                Control      54                                                               ______________________________________                                    

The strains H10, D9 and D20 (comparative) were clearly effective inprotecting fruit from blue mould.

B. Effectiveness of 6 antagonists against grey mould of pear afterholding treated fruit at 0° C. for 10 days

Procedures used were similar to previous examples. Treated and controlfruit were held at 0° C. for 10 days and then transfered to 24° C. for 7days before infection was recorded (Table 11).

                  TABLE 11                                                        ______________________________________                                        Effectiveness of 6 Antagonists Against Grey Mould of Pear After               Holding Treated Fruit at 0° C. for 10 Days                                            Percentage infection                                           Antagonist     after 1 week at 24° C.                                  ______________________________________                                        H9             43                                                             H10             4                                                             D9             12                                                             D20 (Comparative)                                                                            10                                                             D27            31                                                             C.a.           43                                                             Chemical        0                                                             Control        86                                                             ______________________________________                                    

The two examples quoted indicate that three antagonists, viz. H10, D9and D20 (comparative), will retain their activity even after the fruitis held in cold storage for up to 10 days. Further experiments indicatethat the strains H9, D10 and D20 provide protection against infectionfor extended periods such as used for the cold storage of fruit.

EXAMPLE 15

The effectiveness of selected antagonists against transit rot was alsoinvestigated. Transit rot caused by Rhizopus stolonifer on both applesand pears in most countries where pome fruit is grown but is seldomserious. However, when it occurs it can be quite troublesome.

In this experiment D9 and D20 (comparative) were tested with GrannySmith apples. Fruit was wounded and dipped in an aqueous suspension ofthe 3 antagonists and 2 hours later dipped in a suspension ofsporangiospores of R. stolonifer. There were 12 fruit per treatment.Control fruit was treated with the pathogen only. Infection was recordedafter incubating the fruit at 24° C. for 7 days.

Results (Table 12) showed that all 3 antagonists were highly effectivein checking the development of transit rot on apples. Three of thecontrol fruit were almost completely rotten on the 7th day.

                  TABLE 12                                                        ______________________________________                                        Effectiveness of 2 Antagonists Against Transit Rot                            (Rhizopus stolonifer) of Apple                                                           Percentage infection                                               Antagonist after 7 days                                                       ______________________________________                                        D20        0                                                                  D9         0.8                                                                Control    25                                                                 ______________________________________                                    

The above examples show that strains of the yeast species Rhodotorulaglutinis (Fres.) Harrison and Rhodotorula mucilaginosa (Jorg.) Harrison,are effective in the control of microbial diseases of fruit, inparticular, blue and grey mould, Mucor rot of apple and pear and transitrot. It is to be emphasised that the conditions of pathogen infectionare very severe and represent a much higher level of pathogen attackthan experienced under normal conditions. The level of wounding is alsovery much greater than the natural incidence.

This invention has been exemplified with reference to strains H10, D9although it is to be appreciated the invention is not so limited andextends to any strain of the yeast species Rodotorula glutinis (Fres.)Harrison and Rhodotorula mucilaginosa (Jorg.) Harrison, or combinationsthereof. The selected strains H10 and D9 are particularly advantageousdue to their effectiveness in controlling microbial fruit diseases,vigorous growth characteristics and lack of any pathological effects.

This invention in its various aspects also extends to yeast strains ofthe species Geotrichum klebahnii as represented by the strain H9(isolated from a fruit crate) and E. albidus (C.a.) which as describedherein are effective in the control of microbial disease in fruit. Thesestrains have practical disadvantages (when compared with H10 and D9)associated with growth rate and the like which may be overcome bymodification of culture conditions.

The examples have been largely concerned with post-harvest treatment offruit where the application and compositions comprising at least oneyeast strain as described herein can be readily carried out. Theinvention is clearly also applicable to pre-harvest treatment of fruitwhere the compositions of this invention would be applied to the fruitwhen still on the plant.

Yeast strains of the species Rhodotorula glutinis (Fres.) Harrison and,Rhodotorula mucilaginosa (Jorg.) Harrison, are as effective asfungicides currently used in horticulture to prevent microbial diseasesof fruit. Accordingly, the compositions of this invention and methodsinvolving the same provide alternative treatment which do not have theproblems of toxicity posed by fungicides.

Surprisingly, organisms of the species Rhodotorula glutinis (Fres.)Harrison and, Rhodotorula mucilaginosa (Jorg.) Harrison, are notaffected by fungicides, belonging to the benzimidazole and dicarboximidegroups, which are used in field applications for the control ofpre-harvest and post-harvest diseases. Accordingly, compositionsdescribed herein may also contain fungicides in reduced amounts thusovercoming problems of toxicity associated with such fungicides.

The yeast strains referred to in the examples have been shown to survivein a range of temperatures in which fruit is stored and still beeffective.

The mechanism by which the yeast strains of this invention are effectivein the treatment/prevention of microbial disease is currently unclear.They do not appear to produce antibiotics like certain bacteria orfungi. When applied to wounds on fruit they appear to grow until theyhave used all the nutrients available and in this way may be deprivingthe fungal spores of nutrients for their development. When applied aloneto wounds they do not cause any obvious rot or infection.

DEPOSIT DETAILS

All the strains listed hereunder were deposited with the AustralianGovernment Analytical Laboratories, A Budapest Treaty Depository, ofP.O. Box 385, Pymble, 2073, New South Wales, Australia:

    ______________________________________                                        Yeast Strain    Accession No.                                                                             Deposit Date                                      ______________________________________                                        Rhodotorula glutinis H10                                                                      92/15655    6th April, 1992                                   Rhodotorula mucilaginosa D9                                                                   92/15656    6th April, 1992                                   guilliermodii (Cast) Langeron                                                                 92/15657    6th April, 1992                                   and Guerra (teleomorph                                                        Pichia guilliermodii                                                          Wickerman)                                                                    ______________________________________                                    

REFERENCES

The references set out hereunder are specifically incorporated into thespecification by reference.

Barnett, J. A., Payne, R. W. and Yarrow, D., 1983. Yeasts:Characteristics and Identification, Cambridge Univ. Press, Cambridge

Chalutz, E. and Wilson, C. 1990. Postharvest biocontrol of green andblue mould and sour rot of citrus fruit by Debaryomyces Hansenii PlantDisease 74:134-137

Janisiewicz, W. J. 1987. Post-harvest biological control of blue mouldon apples. Phytopathology 77:481-485

Janisiewicz, W. J. 1988. Biocontrol of postharvest diseases of appleswith antagonist mixtures. Phytopathology 78:194-198

King, A. D., Hocking, A. D., and Pitt, J. I. 1979. Dictoran-rose bengalmedium for enumeration and isolation of moulds from foods. Appl.Environ. Microbiol. 37:959-964

Poovia, B. W., Glenn, G. M. and Reddy, A. S. N. 1990. Calcium and fruitsoftening: Physiology and biochemistry. Horticultural Reviews 10:107-152

Wisniewski, M., Wilson, C., Chalutz, E. and Hershberger, W. 1988.Biological control of postharvest diseases of fruit. Inhibition ofBotrytis rot on apple by an antagonistic yeast. Proc. Electron. Microsc.Soc. Am. 46:290-291

I claim:
 1. A composition for the treatment or prevention of microbialdiseases of fruit comprising an effective amount of at least one yeaststrain selected from the species Rhodotorula glutinis (Fres.) Harrison,and Rhodotorula mucilaginosa (Jorg.) Harrison, optionally in associationwith one or more agriculturally acceptable carriers or excipients.
 2. Acomposition according to claim 1 wherein said at least one yeast strainis selected form Rhodotorula glutinis (Fres.) Harrison strain H10deposited with the Australian Government Analytical Laboratory underAccession No. 92/15655; and Rhodotorula mucilaginosa (Jorg.) Harrisonstrain D9 deposited with the Australian Government Analytical Laboratoryunder Accession No. 92/15656; or derivatives or mutants of said strainseffective in the treatment or prevention of microbial disease of fruit.3. A method for the treatment or prevention of microbial disease offruit comprising applying to said fruit an effective amount of acomposition comprising at least one yeast strain selected from thespecies Rhodotorula glutinis (Fres.) Harrison and Rhodotorulamucilaginosa (Jorg.) Harrison, optionally in association with one ormore agriculturally acceptable carriers or excipients.
 4. A methodaccording to claim 3 wherein said composition comprises at least oneyeast strain selected from Rhodotorula glutinis (Fres.) Harrison strainH10 deposited with the Australian Government Analytical Laboratory underAccession No. 92/15656; or derivatives or mutants of said strainseffective in the treatment or prevention of microbial disease of fruit.5. A method for the treatment or prevention of mold or rot of somefruits caused by the organisms Penicillium expansum, Botyris cinerea andMucor piriformis comprising applying to said fruit an effective amountof a composition comprising at least one yeast strain selected from thespecies Rhodotorula glutinis (Fres.) Harrison and Rhodotorulamucilaginosa (Jorg.) Harrison optionally in association with one or moreagriculturally acceptable carriers or excipients.
 6. The method of claim4, wherein Rhodotorula glutinis (Fres.) Harrison strain H10 depositedwith the Australian Government analytical Laboratory under accession as92/15655 is applied to pears in the treatment or prevention of greymold, in a composition comprising at least 1×10⁷ cells/ml.
 7. Acomposition according to claim 1 which additionally comprises a sourceof calcium.
 8. A composition according to claim 7 wherein said source ofcalcium is calcium chloride.
 9. A composition according to claim 1 whichadditionally comprises an antiscald agent.
 10. A composition accordingto claim 9 wherein said antiscald agent is diphenylamine or ethoxyquin.11. A composition according to claim 1 in the form of an aqueoussuspension of said one or more yeast strains.
 12. A compositionaccording to claim 1 comprising at least 1×10⁷ cells/ml.
 13. Acomposition according to claim 1 which is lyophilised.
 14. A compositionaccording to claim 1 for the treatment or prevention of microbialpre-harvest or post-harvest disease of fruit.
 15. A compositionaccording to claim 14 for the treatment or prevention of blue mould,grey mould, and mucor rot of fruit.
 16. A composition according to claim14 for the treatment or prevention of mould or rot of fruit caused bythe organisms P. expansum, Botrytis cinerea, and Mucor piriformis.
 17. Acomposition according to claim 1 for the treatment or prevention ofmicrobial pre-harvest and post-harvest diseases of pome fruit.
 18. Acomposition according to claim 17 wherein said pome fruit is selectedfrom apples and pears.
 19. A composition according to claim 1 for thetreatment or prevention of microbial pre-harvest and post-harvestdisease of stone fruit.
 20. A composition according to claim 19 whereinsaid stone fruit is selected from peaches, nectarines, apricots, plumsand cherries.
 21. A composition according to claim 1 for the treatmentor prevention of microbial pre-harvest and post-harvest diseases ofgrapes.
 22. A composition according to claim 1 for the treatment orprevention of microbial pre-harvest and post-harvest diseases of citrusfruits.
 23. A composition according to claim 22 wherein said citrusfruit is selected from oranges, mandarins, lemons and limes.
 24. Amethod according to claim 3 wherein said composition is an aqueoussuspension of said one or more yeast strains.
 25. A method according toclaim 3 wherein said composition is a composition according to claim 1.26. A method according to claim 3 wherein said composition is applied tofruit by spraying, dipping, drenching or as a mist.
 27. A methodaccording to claim 3 wherein said composition comprises at least 1×10⁷cells/ml.
 28. A method according to claim 3 wherein said composition isapplied to said fruit post-harvest.
 29. A method according to claim 3wherein said composition is applied to fruit pre-harvest.
 30. A methodaccording to claim 3 for the treatment or prevention of microbialpre-harvest and post-harvest diseases of pome fruit.
 31. A methodaccording to claim 30 wherein said pome fruit is selected from applesand pears.
 32. A method according to claim 3 for the treatment orprevention of microbial pre-harvest and post-harvest diseases ofstone-fruit.
 33. A method according to claim 32 wherein said stone fruitis selected from peaches, nectarines, apricots, plums and cherries. 34.A method according to claim 3 for the treatment or prevention ofmicrobial pre-harvest and post-harvest diseases of grapes.
 35. A methodaccording to claim 3 for the treatment or prevention of microbialpre-harvest and post-harvest diseases of citrus fruits.
 36. A methodaccording to claim 35 wherein said citrus fruit is selected fromoranges, mandarins, lemons and limes.
 37. A method according to claim 3for the treatment of blue mould, grey mould, Mucor rot and transit rotof fruit.
 38. A method according to claim 3 which is a method for thetreatment of mould or rot of fruit caused by the organisms P. expansum,Botrytis cinerea and Mucor piriformis.